小鼠胚胎干细胞药物代谢酶CYP3a11基因敲除的实验研究

The knock-out of cytochrome P450 3a11 gene from mouse embryo stem cell

目的研究小鼠胚胎干细胞药物代谢酶CYP3a11基因的敲除。方法根据已知小鼠的CYP3a11基因组DNA序列,从129品系小鼠E14胚胎干细胞基因组DNA中,通过PCR的方法分别获得用于构建基因敲除载体的0.65kb的5'-短臂(short arm,SA)和(3.3+4)kb的3'-长臂(long arm,LA)片段,通过常规分子克隆技术,构建完成针对小鼠药物代谢酶CYP3a11基因的替代型基因敲除载体pCR-CYP3a11_KO,并对载体进行酶切鉴定。将线性化的pCR-CYP3a11_KO载体通过电穿孔技术转入E14胚胎干细胞。G418药物筛选4～6周直至克隆形成,用PCR和Southern blot技术对筛选出的细胞克隆进行鉴定。结果酶切结果表明基因敲除载体pCR-CYP3a11_KO DNA序列正确;转染后的E14胚胎干细胞经过G418筛选后,存活的细胞可形成单克隆集落,经过PCR和Southern blot技术鉴定,其中有5个单克隆被证实CYP3a11基因已被敲除。结论成功构建了小鼠CYP3a11基因敲除载体,并在小鼠E14胚胎干细胞的CYP3a11基因敲除中获得阳性克隆。

Objective To study the knock-out of cytochrome P450 （CYP） 3a11 gene from mouse embryo stem cells. Methods The 0.65 kb 5＇ arm （short arm,SA） and the （3.3 ＋ 4） kb 3＇ arm （long arm,LA） of the mouse CYP3a11 gene knock-out vector were amplified by PCR according to the known sequence of genomic gene. The LA,PGK neo,SA and tk were sequentially cloned into pCR2.1-TOPO vector to construct the knock-out vector pCR-CYP3a11_KO. The pCR-CYP3a11_KO was linearized and transfected into mouse embryo stem cell line E14 by electroporation. After 5-6 weeks of G418 selection,picked the cell colonies and checked them by PCR and southern blot. Results The sequence of the knock-out vector pCR-CYP3a11_KO was confirmed according to the digestive results of the restriction endonucleases. The transfected E14 cells could form clonies after selection of G418 and Ganc. The CYP3a11 gene of five E14 cell colonies was verified being knockouted. Conclusion The results indicated that the drug-metabolizing enzyme CYP3a11 gene knock-out vector had been constructed and the mouse E14 embryo stem cell colonies with knock outed CYP3a11 gene were gained.